Combination therapies using HDAC inhibitors

ABSTRACT

The present invention pertains to a method for treating cancer, such as lung cancer, multiple myeloma, lymphoma, and epithelial ovarian cancer, comprising the administration to a patient in need thereof a first amount or dose of a histone deacetylase (HDAC) inhibitor, such as PXD-101, and a second amount or dose of another chemotherapeutic agent, such as dexamethasone or 5-fluorouracil, or an epidermal growth factor receptor (EGFR) inhibitor, such as TarcevaÚ, wherein the first and second amounts or doses together comprise a therapeutically effective amount.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/554,261, filed Nov. 26, 2014, which is a continuationapplication of U.S. patent application Ser. No. 11/815,372, filed Oct.9, 2007, now abandoned, which is a National Phase Entry Under 35 U.S.C.§ 371 of International Application Serial No. PCT/GB2006/000391, filedFeb. 3, 2006, which claims priority from U.S. Prov. Application Ser. No.60/649,991, filed Feb. 3, 2005 and U.S. Prov. Application Ser. No.60/735,662, filed Nov. 10, 2005, the disclosures of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to methods for treating cancer. Morespecifically, the present invention relates to methods for treatingcancer, such as lung cancer, multiple myeloma, lymphoma, and epithelialovarian cancer, in a subject, said methods comprising administering to apatient in need thereof a therapeutically effective amount of a histonedeacetylase inhibitor (e.g., PXD-101 or its analogs), and a secondamount or dose of a (i.e., another) chemotherapeutic agent (e.g.,dexamethasone or 5-fluorouracil) and/or an epidermal growth factorreceptor (EGFR) inhibitor (e.g., Tarceva®).

BACKGROUND OF THE INVENTION

A number of patents and publications are cited herein in order to morefully describe and disclose the invention and the state of the art towhich the invention pertains. Each of these references is incorporatedherein by reference in its entirety into the present disclosure, to thesame extent as if each individual reference was specifically andindividually indicated to be incorporated by reference.

Throughout this specification, including the claims which follow, unlessthe context requires otherwise, the word “comprise,” and variations suchas “comprises” and “comprising,” will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

As used herein, “a,” “an” and “the” include singular and pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “an active agent” or “a pharmacologically activeagent” includes a single active agent as well a two or more differentactive agents in combination, reference to “a carrier” includes mixturesof two or more carriers as well as a single carrier, and the like.

Ranges are often expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by the use of the antecedent “about,” itwill be understood that the particular value forms another embodiment.

Multiple Myeloma

Multiple myeloma is a disseminated malignancy of plasma cells thataffects approximately 14,600 new patients each year in the UnitedStates. The etiology of this rare blood disease, affecting mainly themiddle-aged to elderly population, is largely unknown although geneticpredisposition and environmental factors have been implicated. Fromonset, malignant plasma cells arising from clonal expansion accumulatein the bone marrow, producing abnormally high levels of immunoglobulins.Multiple myeloma is difficult to diagnose early because there may be nosymptoms in early stage. Bone pain especially secondary to compressionfractures of the ribs or vertebrae is the most common symptom.

Dexamethasone is a commonly used regimen for first-line treatment ofthis disease. More recently, combinations of vincristine, doxorubicin,and dexamethasone (VAD) have been used to treat multiple myeloma.However, these are not effective long-term treatments. Dexamethasonetreatment has a response rate of approximately 25-35%.

In many patients, high-dose chemotherapy supported by autologous stemcell transplantation (ASCT) may prolong event-free survival if theprocedure is performed within 12 months of initial diagnosis. Howeveralmost all patients receiving high-dose chemotherapy and an autologousperipheral stem cell transplant will ultimately relapse.

Lymphoma

Despite years of research into the development of new methods oftreatment, cancers of the lymphatic system, or lymphomas, remain quitecommon. For example, more than 60,000 people in the United States arediagnosed with lymphoma each year, including more than 55,000 cases ofnon-Hodgkin's Lymphoma (NHL), and these numbers are constantlyincreasing. In addition, the prognosis for those affected by thesediseases is often poor, as the survival rates for lymphoma patientsremain low. Clearly, new methods for treating these diseases are needed.

While traditional treatments for lymphoma typically depend on the typeof lymphoma as well as the medical history of the patient, first-linetreatment for many lymphomas typically includes chemotherapy. Suchchemotherapy will often entail the administration of a “cocktail” ofcompounds, e.g., the formulation CHOP, which includes cyclophosphamide,doxorubicin, vincristine, and prednisone. In addition, certainfirst-line cancer treatments also include other forms of cancer therapy,such as radiation therapy.

In many cases, patients respond initially to such first-line treatments,but subsequently suffer a relapse, i.e., a tumor reappears or resumesgrowing. Following one such relapse, patients are often treated withfurther chemotherapy, e.g., with CHOP or with other formulations, or, insome cases, the patients are treated with other procedures such as bonemarrow transplantation. Again, in many cases, patients initially respondto such additional treatments, but subsequently suffer another relapse.In general, the more relapses a patient suffers, the less agreementthere is in the art concerning optimal subsequent treatment. In othercases, a patient fails to respond at all to a treatment, even initially,and is thus said to have a refractory cancer. In such cases as well,little agreement exists in the art regarding optimal subsequenttreatment.

Prostate Cancer

Prostate cancer is the most common cancer leading to death among UnitedStates men. Its incidence has increased considerably over the past twodecades. This cancer is often initially responsive to hormone treatment,but then frequently progresses into a hormone-insensitive state that isnot easily treated. Further, once the cancer spreads or metastasizesfrom the prostate gland to other tissues such as bone marrow, the cancerusually cannot be cured by presently available treatments, which areeither too toxic or cannot reach the metastases. There is a dire needfor new strategies to treat prostate cancers, including metastasizedprostate cancers, with low toxicity.

Ovarian Cancer

Ovarian cancer is the fourth leading cause of cancer deaths among womenin the United States and causes more deaths than all other gynecologicmalignancies combined.

In the United States, a woman's lifetime risk of developing ovariancancer is 1 in 70. In 1992, about 21,000 cases of ovarian cancer werereported, and about 13,000 women died from the disease. See, e.g.,Chapter 321, Ovarian Cancer, Harrison's Principles of Internal Medicine,13th ed., Isselbacher et al., eds., McGraw-Hill, New York (1994), pages1853-1858; and American Cancer Society Statistics, Cancer J. Clinicians,45:30 (1995). Epithelial ovarian cancer, the most common ovarian cancer,has a distinctive pattern of spread in which cancer cells migratethroughout the peritoneal cavity to produce multiple metastatic nodulesin the visceral and parietal peritoneum and the hemidiaphragms. Inaddition, metastasis can occur to distant sites such as the liver, lungand brain. Early stage ovarian cancer is often asymptomatic and isdetected coincidentally by palpating an ovarian mass on pelvicexamination. In premenopausal patients, about 95% of these masses arebenign. Even after menopause, 70% of masses are benign but detection ofany enlargement requires evaluation to rule out malignancy. Inpostmenopausal women with a pelvic mass, a markedly elevated serumCA-125 level of greater than 65 U/mL indicates malignancy with a 96%positive predictive value. See, e.g., Chapter 321, Ovarian Cancer,Harrison's Principles of Internal Medicine, 13th ed., Isselbacher etal., eds., McGraw-Hill, New York (1994).

Epithelial ovarian cancer is seldom encountered in women less than 35years of age. Its incidence increases sharply with advancing age andpeaks at ages 75 to 80, with the median age being 60 years. The singlemost important risk factor for this cancer is a strong family history ofbreast or ovarian cancer. Oncogenes associated with ovarian cancersinclude the HER-2/neu (c-erbB-2) oncogene, which is overexpressed in athird of ovarian cancers, the fms oncogene, and abnormalities in the p53gene, which are seen in about half of ovarian cancers. A number ofenvironmental factors have also been associated with a higher risk ofepithelial ovarian cancer, including a high fat diet and intake oflactose in subjects with relatively low tissue levels ofgalactose-1-phosphate uridyl transferase.

The internationally accepted first-line chemotherapy for advancedepithelial ovarian cancer is the combination of carboplatin andpaclitaxel. Typical results are median progression-free survival (PFS)of 17-20 months and median survival of 3-5 years. Second-line treatmentis determined by duration of remission. If relapse occurs within 6months of the last treatment, patients are considered “platinumresistant.” Re-treatment with a carboplatin/paclitaxel regimen in thesepatients is associated with a low response rate (15%) of short duration(3-6 months), and a median survival of approximately 12 months. For thispatient population, there is a need for a more effective therapy.

Lung Cancer

Lung cancer is the leading cause of cancer mortality in the UnitedStates (“Cancer Facts and FIGS. 2003,” American Cancer Society). Lungcancer is particularly insidious because symptoms of early-stage,localized disease are nonspecific and are frequently attributed to theeffects of smoking. By the time the patient seeks medical attention, thedisease is usually advanced so that complete surgical resection ispossible in less than 30% of all cases, and the overall 5-year survivalrate in less than 15%. See, e.g., “Cancer of the Lung: Cancer Screeningand Early Detection,” in Cancer Medicine, 5th Edition, Bast et al. eds.,B.C. Decker Inc., Hamilton, Ontario, Canada.

Surgery and radiotherapy may be curative if a cancer is found early, butcurrent drug therapies for metastatic disease are mostly palliative andseldom offer a long-term cure. Even with the new chemotherapies enteringthe market, improvement in patient survival is measured in months ratherthan in years, and the need continues for new drugs effective both incombination with existing agents as first line therapy and as second andthird line therapies in treatment of resistant tumors.

HDAC Inhibitors

Histones are major protein components of chromatin. The regulation ofchromatin structure is emerging as a central mechanism for the controlof gene expression. As a general paradigm, acetylation of the ϵ-aminogroups of lysine residues in the amino-terminal tails of nucleosomalhistones is associated with transcriptional activation, whiledeacetylation is associated condensation of chromatin andtranscriptional repression. Acetylation and deacetylation of histones iscontrolled by the enzymatic activity of histone acetyltransferases(HATs) and histone deacetylases (HDACs). Several transcription factorsincluding p53 and GATA-1 have also been shown to be substrates forHDACs.

Prototypical HDAC inhibitors, such as the natural products trichostatinA (TSA) and suberoyl hydroxamic acid (SAHA), induce the expression ofgenes associated with cell cycle arrest and tumor suppression.Phenotypic changes induced by HDAC inhibitors include G1, and G2/M cellcycle arrest and apoptosis in tumor cells. Antitumor activity has beendemonstrated in vivo in animal models with a number of HDAC inhibitors,including PXD-101.

PXD-101 is a potent HDAC inhibitor that belongs to the hydroxymate-typeof histone deacetylase inhibitors, which for various members of thegroup has shown pronounced in vitro and in vivo (pre-clinical and earlyclinical trials) activity against myeloma and lymphoma.

SUMMARY OF THE INVENTION

The present invention is based upon the discovery that histonedeacetylase (HDAC) inhibitors, such as PXD-101, can be used incombination with chemotherapeutic agents and/or epidermal growth factorreceptor (EGFR) inhibitors to provide therapeutically effectiveanticancer effects. Furthermore, an unexpected synergistic interactionbetween the HDAC inhibitor and the chemotherapeutic agent or the EGFRinhibitor results, wherein the combined effect is greater than theadditive effect resulting from administration of the two treatments eachat a therapeutic dose.

Thus, one aspect of the present invention relates to a method for thetreatment of cancer, wherein the method comprises administering to apatient in need thereof, a first amount or dose of a histone deacetylase(HDAC) inhibitor, such as PXD-101, and a second amount or dose of a(i.e., another) chemotherapeutic agent, such as dexamethasone or5-fluorouracil, or an epidermal growth factor receptor (EGFR) inhibitor,such as Tarceva®. The first and second amounts or doses togethercomprise a therapeutically effective amount.

Another aspect of the present invention pertains to use of a histonedeacetylase (HDAC) inhibitor, such as PXD101, in the manufacture of amedicament for the treatment of cancer, wherein said treatment comprisestreatment with (i) said histone deacetylase inhibitor and (ii) anotherchemotherapeutic agent, such as dexamethasone or 5-fluorouracil, or anepidermal growth factor receptor (EGFR) inhibitor, such as Tarceva®.

Another aspect of the present invention pertains to use of anotherchemotherapeutic agent, such as dexamethasone or 5-fluorouracil, or anepidermal growth factor receptor (EGFR) inhibitor, such as Tarceva®, inthe manufacture of a medicament for the treatment of cancer, whereinsaid treatment comprises treatment with (i) a histone deacetylase (HDAC)inhibitor, such as PXD-101, and (ii) said another chemotherapeutic agentor epidermal growth factor receptor (EGFR) inhibitor.

The treatment procedures described herein can take place sequentially inany order, simultaneously, or a combination thereof. For example, thefirst treatment procedure, administration of a histone deacetylase(HDAC) inhibitor, can take place prior to the second treatmentprocedure, administration of a (i.e., another) chemotherapeutic agent ora epidermal growth factor receptor (EGFR) inhibitor; after the secondtreatment procedure; at the same time as the second treatment procedure;or a combination thereof. For example, a total treatment period can bedecided for the HDAC inhibitor. The second treatment can be administeredprior to onset of treatment with the HDAC inhibitor, or followingtreatment with the HDAC inhibitor. In addition, second treatment can beadministered during the period of HDAC inhibitor administration, butdoes not need to occur over the entire HDAC inhibitor treatment period.

The combination therapy can provide a therapeutic advantage in view ofthe differential toxicity associated with the two treatment modalities.More specifically, treatment with HDAC inhibitors can lead tohematologic toxicity, whereas chemotherapy treatments can be toxic totissue adjacent the tissue site. As such, this differential toxicity canpermit each treatment to be administered at its therapeutic dose,without increasing patient morbidity. Surprisingly however, thetherapeutic effects achieved as a result of the combination treatmentare enhanced or synergistic, for example, significantly better thanadditive therapeutic effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a graph (% inhibition vs. Log M DEX) demonstrating theresults of a combination study involving PXD-101 and dexamethasone on amyeloma cell line (i.e., U266 cells): PXD-101 pre-incubation, followedby PXD-101+dexamethasone). The lower curve is DEX only and the uppercurve is PXD-101+DEX.

FIG. 2 depicts a graph (combination index vs. combination number)showing the synergy of PXD-101 and 5-FU in a WST-1 anti-proliferativeassay as calculated by CalcuSyn® (HCT116, 24 hour PXD-101+48 hour 5-FUALONE, Combination). See also Data Annex 5.

FIG. 3 depicts a graph (combination index vs. combination number)showing the synergy of PXD-101 and 5-FU in a clonogenic assay ascalculated by CalcuSyn® (HCT116 PXD-101/5-FU 24 h co-incubation).

FIG. 4 depicts a graph (tumor volume (cm³) vs. days) representing thechemo-sensitizing effect of PXD-101 (Control; 5-FU, 15 mg/kg; PXD-101,100 mg/kg; PXD-101/5-FU, 100/15 mg/kg).

FIG. 5 depicts two bar graphs (in vitro growth inhibition (%)) for(top): PXD-101 (0.25 μM), Tarceva® (0.32 μM), and the combination(PXD-101/Tarceva® on Calu03, 72 hour co-treatment); and (bottom);PXD-101 (0.35 μM), Tarceva® (0.04 μM), and the combination(PXD-101/Tarceva® on HCC-4006, 72 hour co-treatment).

FIG. 6 depicts two bar graphs (in vitro growth inhibition (%)) for(top): PXD-101 (1.8 μM), Alimta® (0.16 μM), and the combination(PXD-101/Alimta® on A549, 24 hour Alimta®, 48 hour co-treatment); and(bottom): PXD-101 (1.8 μM), Alimta® (0.04 μM), and the combination(PXD-101/Alimta® on NCI-H460, 24 hour Alimta®, 48 hour co-treatment).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for the treatment of cancer.The methods comprise administering to a patient in need thereof a firstamount or dose of a histone deacetylase (HDAC) inhibitor, such asPXD-101, and a second amount or dose of a (i.e., another)chemotherapeutic agent, such as dexamethasone or 5-fluorouracil, or anepidermal growth factor receptor (EGFR) inhibitor, such as Tarceva®. Thefirst and second amounts or doses together comprise a therapeuticallyeffective amount.

(It is intended that histone deacetylase (HDAC) inhibitor is differentfrom the chemotherapeutic agent or an EGFR inhibitor; that is, that thecombination therapy is treatment with at least two different agents.)

In one embodiment, the second amount or dose is a second amount or doseof a (i.e., another) chemotherapeutic agent.

In one embodiment, the second amount or dose is a second amount or doseof an epidermal growth factor receptor (EGFR) inhibitor.

In one embodiment, the second amount or dose is a second amount or doseof a compound selected from: Cisplatin, 5-Fluorouracil, Oxaliplatin,Topotecan, Gemcitabine, Docetaxel, Doxorubicin, Tamoxifen,Dexamethasone, 5-Azacytidine, Chlorambucil, Fludarabine, Tarceva®,Alimta®, Melphalan, and pharmaceutically acceptable salts and solvatesthereof.

In one embodiment, the method provides an anticancer effect which issynergistic.

As used herein, the terms “treatment of cancer” and “cancer treatment”refer to partially or totally inhibiting, delaying, or preventing theprogression of cancer, including cancer metastasis; inhibiting,delaying, or preventing the recurrence of cancer, including cancermetastasis; or preventing the onset or development of cancer(chemoprevention) in a mammal, for example a human.

As used herein, the term “therapeutically effective amount” is intendedto qualify the combined amount of the first and second treatments in thecombination therapy. The combined amount will achieve the desiredbiological response. In the present invention, the desired biologicalresponse is treatment of cancer, i.e., partial or total inhibition,delay, or prevention of the progression of cancer, including cancermetastasis; inhibition, delay, or prevention of the recurrence ofcancer, including cancer metastasis; or the prevention of the onset ordevelopment of cancer (chemoprevention) in a mammal, for example ahuman.

The combination therapy of the present invention is suitable for use inthe treatment of a wide variety of cancers. As used herein, the term“cancer” refers to tumors, neoplasms, carcinomas, sarcomas, leukemias,lymphomas, and the like. For example, cancers include, but are notlimited to, leukemias and lymphomas such as cutaneous T-cell lymphoma(CTCL), noncutaneous peripheral T-cell lymphoma, lymphomas associatedwith human T-cell lymphotropic virus (HTLV), for example, adult T-cellleukemia/lymphoma (ATLL), acute lymphocytic leukemia, acutenonlymphocytic leukemias, chronic lymphocytic leukemia, chronicmyelogenous leukemia, Hodgkin's Disease, non-Hodgkin's lymphomas, andmultiple myeloma, childhood solid tumors such as brain tumors,neuroblastoma, retinoblastoma, Wilms' Tumor, bone tumors, andsoft-tissue sarcomas, common solid tumors of adults such as head andneck cancers (e.g., oral, laryngeal, and esophageal), genitourinarycancers (e.g., prostate, bladder, renal, uterine, ovarian, testicular,rectal, and colon), lung cancer, breast cancer, pancreatic cancer,melanoma and other skin cancers, stomach cancer, brain cancer, livercancer and thyroid cancer.

In one embodiment, the cancer to be treated by the methods disclosedherein is lung cancer.

In one embodiment, the cancer to be treated by the methods disclosedherein is multiple myeloma.

In one embodiment, the cancer to be treated by the methods disclosedherein is lymphoma.

In one embodiment, the cancer to be treated by the methods disclosedherein is epithelial ovarian cancer.

Histone Deacetylases and Histone Deacetylase Inhibitors

Histone deacetylases (HDACs) are involved in the reversible acetylationof histone and non-histone proteins (p53, tubulin, and varioustranscription factors). Mammalian HDACs have been ordered into threeclasses based upon their similarity to known yeast factors. Class IHDACs (HDACs 1, 2, 3 and 8) bear similarity to the yeast RPD3 protein,are located in the nucleus and are found in complexes associated withtranscriptional co-repressors. Class II HDACs (HDACs 4, 5, 6, 7 and 9)are similar to the yeast HDA1 protein, and have both nuclear andcytoplasmic subcellular localization. Class III HDACs form astructurally distant class of NAD dependent enzymes that are related tothe yeast SIR2 proteins.

Compounds that have been shown to inhibit HDAC activity fall into fivestructurally diverse classes: (1) hydoxamic acids; (2) cyclictetrapeptides; (3) aliphatic acids; (4) benzamides; and (5)electrophillic ketones.

Hydroxamic acids were among the first HDAC inhibitors identified andthese agents helped define the model pharmacophore for HDAC inhibitors.The linker domain of these agents is comprised of linear or cyclicstructures, either saturated or unsaturated, and the surface recognitiondomain is generally a hydrophobic group, most often aromatic. Phase Iand II clinical trials are currently on-going for several hydoxamicacid-based HDAC inhibitors, including PXD-101.

PXD-101 is a highly potent HDAC inhibitor that blocks proliferation ofdiverse tumor cell lines at low micromolar potency (IC₅₀ 0.08-2.43 μM)and HDAC enzyme activity (IC₅₀ 9-110 nM). In xenograft models, PXD-101slows tumor growth in a dose dependent manner and is particularly activein leukemic mouse models. In addition, PXD-101 causes cell cycle arrestand apoptosis in rapidly proliferating cells. Thus, hydoxamic acid-basedHDAC inhibitors are suitable for use in the present invention.

In one embodiment, the histone deacetylase (HDAC) inhibitor is selectedfrom compounds represented by the following formula:

wherein:

-   -   A is an unsubstituted phenyl group;    -   Q¹ is a covalent bond, a C₁₋₇alkylene group, or a C₁₋₇alkenylene        group;    -   J is:

-   -   R¹ is hydrogen, C₁₋₇alkyl, C₃₋₂₀heterocyclyl, C₅₋₂₀aryl, or        C₅₋₂₀aryl-C₁₋₇alkyl; and    -   Q² is:

and pharmaceutically acceptable salts and solvates thereof.

In one embodiment, Q¹ is a covalent bond or a C₁₋₇alkylene group.

In one embodiment, Q¹ is a covalent bond.

In one embodiment, Q¹ is a C₁₋₇alkylene group.

In one embodiment, Q¹ is —CH₂—, —CH₂CH₂—, —CH₂CH(CH₃)—, —CH₂CH(CH₂CH₃)—,—CH₂CH₂CH₂—, or —CH₂CH₂CH₂CH₂—.

In one embodiment, Q¹ is a C₁₋₇alkenylene group.

In one embodiment, Q¹ is —CH═CH— or —CH═CH—CH₂—.

In one embodiment, R¹ is hydrogen, C₁₋₇alkyl, C₃₋₂₀heterocyclyl, orC₅₋₂₀aryl.

In one embodiment, R¹ is hydrogen.

In one embodiment, R¹ is C₁₋₇alkyl.

In one embodiment, R¹ is -Me, -Et, -nPr, -iPr, -nBu, -iBu, -sBu, or-tBu.

In one embodiment, R¹ is -Me, -nBu, or -iBu

In one embodiment, R¹ is C₃₋₂₀heterocyclyl.

In one embodiment, R¹ is C₃₋₂₀aryl.

In one embodiment, R¹ is phenyl.

In one embodiment, R¹ is C₅₋₂₀aryl-C₁₋₇alkyl.

In one embodiment, R¹ is benzyl.

In one embodiment, the histone deacetylase (HDAC) inhibitor is selectedfrom the following compounds, and pharmaceutically acceptable salts orsolvates thereof

In one embodiment, the histone deacetylase (HDAC) inhibitor is selectedfrom PXD-101, which is represented by the following formula, andpharmaceutically acceptable salts or solvates thereof:

Other histone deacetylase (HDAC) inhibitors suitable for use in thepresent invention include the compounds disclosed in U.S. patentapplication Ser. Nos. 10/381,790; 10/381,794; and Ser. No. 10/381,791;the contents of each of which are hereby incorporated by reference intheir entirety.

Stereoisomers

This invention is intended to encompass, in addition to the use of theabove listed compounds, the use of stereoisomers of such compounds andmixtures thereof.

Many organic compounds exist in optically active forms having theability to rotate the plane of plane-polarized light. In describing anoptically active compound, the prefixes D and L or R and S are used todenote the absolute configuration of the molecule about its chiralcenter(s). The prefixes d and l or (+) and (−) are employed to designatethe sign of rotation of plane-polarized light by the compound, with (−)or meaning that the compound is levorotatory. A compound prefixed with(+) or d is dextrorotatory. For a given chemical structure, thesecompounds, called stereoisomers, are identical except that they arenon-superimposable mirror images of one another. A specific stereoisomercan also be referred to as art enantiomer, and a mixture of such isomersis often called an enantiomeric mixture. A 50:50 (by mole) mixture ofenantiomers is referred to as a racemic mixture. Many of the compoundsdescribed herein can have one or more chiral centers and therefore canexist in different enantiomeric forms. If desired, a chiral carbon atomcan be designated with an asterisk (*). When bonds to the chiral carbonatom are depicted as straight lines in the formulas of the invention, itis understood that both the (R) and (S) configurations of the chiralcarbon atom, and hence both enantiomers and mixtures thereof, areembraced within the formula. As is used in the art, when it is desiredto specify the absolute configuration about a chiral carbon, one of thebonds to the chiral carbon can be depicted as a wedge (bonds to atomsabove the plane) and the other can be depicted as a series or wedge ofshort parallel lines is (bonds to atoms below the plane). TheCahn-Inglod-Prelog system can be used to assign the (R) or (S)configuration to a chiral carbon.

When the HDAC inhibitors described herein contain one chiral center, thecompounds exist in two enantiomeric forms. The enantiomers can beresolved by methods known to those skilled in the art, for example byformation of diastereoisomer salts which can be separated, for example,by crystallization (See, e.g., CRC Handbook of Optical Resolutions viaDiastereomeric Salt Formation by David Kozma (CRC Press, 2001));formation of diastereoisomer derivatives or complexes which can beseparated, for example, by crystallization, gas-liquid or liquidchromatography; selective reaction of one enantiomer with anenantiomer-specific reagent, for example enzymatic esterification; orgas-liquid or liquid chromatography in a chiral environment, for exampleon a chiral support for example silica with a bound chiral ligand or inthe presence of a chiral solvent. It will be appreciated that where thedesired enantiomer is converted into another chemical entity by one ofthe separation procedures described above, a further step is required toliberate the desired enantiomeric form. Alternatively, specificenantiomers can be synthesized by asymmetric synthesis using opticallyactive reagents, substrates, catalysts or solvents, or by converting oneenantiomer into the other by asymmetric transformation. This inventionis intended to encompass, in addition to the use of the above listedcompounds, the use of each enantiomer and mixtures of enantiomers, suchas the specific 50:50 (by mole) mixture referred to as a racemicmixture.

Designation of a specific absolute configuration at a chiral carbon ofthe compounds is understood to mean that the designated enantiomericform of the compounds is in enantiomeric excess (ee) or in other wordsis substantially free from the other enantiomer. For example, the “R”forms of the compounds are substantially free from the “S” forms of thecompounds and are; thus, in enantiomeric excess of the “S” forms.Conversely, “S” forms of the compounds are substantially free of “R”forms of the compounds and are, thus, in enantiomeric excess of the “R”forms. Enantiomeric excess, as used herein, is the presence of aparticular enantiomer at greater than 50%. For example, the enantiomericexcess can be about 60% or more, such as about 70% or more, for exampleabout 80% or more, such as about 90% or more. In a particular embodimentwhen a specific absolute configuration is designated, the enantiomericexcess of depicted compounds is at least about 90%. In a more particularembodiment, the enantiomeric excess of the compounds is at least about95%, such as at least about 97.5%, for example, at least 99%enantiomeric excess.

When a compound has two or more chiral carbon atoms it can have morethan two optical isomers and can exist in diastereoisomeric forms. Forexample, when there are two chiral carbon atoms, the compound can haveup to 4 optical isomers and 2 pairs of enantiomers ((S,S)/(R,R) and(R,S)/(S,R)). The pairs of enantiomers (e.g., (S,S)/(R,R)) are mirrorimage stereoisomers of one another. The stereoisomers which are notmirror-images (e.g., (S,S) and (R,S)) are diastereomers. Thediastereoisomer pairs can be separated by methods known to those skilledin the art, for example chromatography or crystallization and theindividual enantiomers within each pair can be separated as describedabove. This invention is intended to encompass, in addition to the useof above listed compounds, the use of each diastereoisomer of suchcompounds and mixtures thereof.

Salts and Solvates

The active compounds disclosed herein can, as noted above, be preparedin the form of their pharmaceutically acceptable salts. Pharmaceuticallyacceptable salts are salts that retain the desired biological activityof the parent compound and do not impart undesired toxicologicaleffects. Examples of pharmaceutically acceptable salts are discussed inBerge et al., 1977, “Pharmaceutically Acceptable Salts,” J. Pharm. Sci.,Vol. 66, pp. 1-19.

The active compounds disclosed can, as noted above, be prepared in theform of their solvates. The term “solvate” is used herein in theconventional sense to refer to a complex of solute (e.g., activecompound, salt of active compound) and solvent. If the solvent is water,the solvate may be conveniently referred to as a hydrate, for example, ahemihydrate, monohydrate, dihydrate, trihydrate, tetrahydrate, and thelike.

Prodrugs

This invention is intended to encompass, in addition to the use of theabove listed compounds, the use of pro-drugs of the compound (e.g., HDACinhibitors) disclosed herein. A prodrug of any of the compounds can bemade using well known pharmacological techniques.

Homologs and Analogs

This invention is intended to encompass, in addition to the use of theabove listed compounds, the use of homologs and analogs of suchcompounds. In this context, homologs are molecules having substantialstructural similarities to the above-described compounds and analogs aremolecules having substantial biological similarities regardless ofstructural similarities.

Chemotherapeutic Agents

The chemotherapeutic agents suitable for use in the present inventioninclude one or more other anti-tumor substances, for example thoseselected from, for example, mitotic inhibitors, for example vinblastine;alkylating agents, for example cisplatin, carboplatin, andcyclophosphamide; inhibitors of microtubule assembly, like paclitaxel orother taxanes; antimetabolites, for example 5-fluorouracil,capecitabine, cytosine arabinoside, and hydroxyurea, or, for example,intercalating antibiotics, for example, adriamycin and bleomycin;immunostimulants, for example trastuzumab; DNA synthesis inhibitors, forexample, gemcitabine; enzymes, for example asparaginase; topoisomeraseinhibitors, for example etoposide; biological response modifiers, forexample interferon; and anti-hormones, for example, antioestrogens suchas tamoxifen or, for example, antiandrogens, such as(4′-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3′-(trifluoromethyl)-propionanilide,or other therapeutic agents and principles as described in, for example,DeVita, V. T., Jr., Hellmann, S., Rosenberg, S. A.; in: Cancer:Principles & Practice of Oncology, 5th ed., Lippincott-Raven Publishers(1997).

In one embodiment, the chemotherapeutic agent is dexamethasone.

In one embodiment, the chemotherapeutic agent is 5-fluorouracil.

EGFR Inhibitors

As used herein, the term “EGFR inhibitor” refers to a molecule havingthe ability to inhibit a biological function of a native epidermalgrowth factor receptor (EGFR). Accordingly, the term “inhibitor” isdefined in the context of the biological role of EGFR. While preferredinhibitors herein specifically interact with (e.g., bind to) an EGFR,molecules that inhibit an EGFR biological activity by interacting withother members of the EGFR signal transduction pathway are alsospecifically included within this definition. A preferred EGFRbiological activity inhibited by an EGFR inhibitor is associated withthe development, growth, or spread of a tumor. EGFR inhibitors, withoutlimitation, include peptides, non-peptide small molecules, antibodies,antibody fragments, antisense molecules, and oligonucleotide decoys.

In one embodiment, the EGFR inhibitor is Tarceva®.

Some Preferred Chemotherapeutic Agents and EGFR Inhibitors

Examples of some preferred chemotherapeutic agents and EGFR inhibitorsinclude: Cisplatin, 5-Fluorouracil, Oxaliplatin, Topotecan, Gemcitabine,Docetaxel, Doxorubicin, Tamoxifen, Dexamethasone, 5-Azacytidine,Chlorambucil, Fludarabine, Tarceva®, Alimta®, Melphalan.

Thus, in one embodiment, the treatment comprises treatment with (i) ahistone deacetylase (HDAC) inhibitor, such as PXD-101, and (ii) acompound selected from: Cisplatin, 5-Fluorouracil, Oxaliplatin,Topotecan, Gemcitabine, Docetaxel, Doxorubicin, Tamoxifen,Dexamethasone, 5-Azacytidine, Chlorambucil, Fludarabine, Tarceva®,Alimta®, Melphalan, and pharmaceutically acceptable salts and solvatesthereof.

Modes and Doses of Administration

The methods of treatment of the present invention comprise administeringto a patient in need thereof a first amount or dose of a histonedeacetylase (HDAC) inhibitor, such as PXD-101, in a first treatmentprocedure, and a second amount or dose of a (i.e., another)chemotherapeutic agent, such as dexamethasone or 5-fluorouracil, or anepidermal growth factor receptor (EGFR) inhibitor, such as Tarceva®, ina second treatment procedure. The first and second amounts togethercomprise a therapeutically effective amount. In some embodiments, thecombination therapy results in a synergistic effect.

The term “patient”, as used herein, refers to the recipient of thetreatment. Mammalian and non-mammalian patients are included. In oneembodiment, the patient is a mammal. In one embodiment, the patient is arodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine (e.g., amouse), a lagomorph (e.g., a rabbit), avian (e.g., a bird), canine(e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), porcine(e.g., a pig), ovine (e.g., a sheep), bovine (e.g., a cow), caprine(e.g., a goat), a primate, simian (e.g., a monkey or ape), a monkey(e.g., marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orangutang,gibbon), or a human. In one embodiment, the patient is human, canine,feline, murine, bovine, ovine, porcine, or caprine.

In one embodiment, the patient is a human.

Administration of HDAC Inhibitor

The histone deacetylase (HDAC) inhibitors of the invention can beadministered in oral forms such as tablets, capsules (each of whichincludes sustained release or timed release formulations), pills,powders, granules, elixers, tinctures, suspensions, syrups, andemulsions.

Likewise, the histone deacetylase (HDAC) inhibitors can be administeredin intravenous (bolus or infusion), intraperitoneal, subcutaneous, orintramuscular form, all using forms well known to those of ordinaryskill in the pharmaceutical arts.

The histone deacetylase (HDAC) inhibitors can be administered in theform of a depot injection or implant preparation which can be formulatedin such a manner as to permit a sustained release of the activeingredient. The active ingredient can be compressed into pellets orsmall cylinders and implanted subcutaneously or intramuscularly as depotinjections or implants. Implants can employ inert materials such asbiodegradable polymers or synthetic silicones, for example, Silastic®,silicone rubber, or other polymers, e.g., manufactured by theDow-Corning Corporation.

The HDAC inhibitor can also be administered in the form of liposomedelivery systems, such as small unilamellar vesicles, large unilamellarvesicles and multilamellar vesicles. Liposomes can be formed from avariety of phospholipids, such as cholesterol, stearylamine, orphosphatidylcholines.

The HDAC inhibitors can also be delivered by the use of monoclonalantibodies as individual carriers to which the compound molecules arecoupled.

The HDAC inhibitors can also be prepared with soluble polymers astargetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer,polyhydroxy-propyl-methacrylamide-phenol,polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, the HDAC inhibitorscan be prepared with biodegradable polymers useful in achievingcontrolled release of a drug, for example, polylactic acid, polyglycolicacid, copolymers of polylactic and polyglycolic acid, polyepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacrylates, and cross linked or amphipathicblock copolymers of hydrogels.

Dosage Regimens

The dosage regimen utilizing the HDAC inhibitors can be selected inaccordance with a variety of factors including type, species, age,weight, sex and the type of cancer being treated; the severity (i.e.,stage) of the cancer to be treated; the route of administration; therenal and hepatic function of the patient; and the particular compoundor salt thereof employed. An ordinarily skilled physician orveterinarian can readily determine and prescribe the effective amount ofthe drug required to treat, for example, to prevent, inhibit (fully orpartially) or arrest the progress of the disease.

Dosage Rates and Formulations

Oral dosages of histone deacetylase (HDAC) inhibitors, when used totreat the desired cancer (e.g., in a human), can range between about 2mg to about 2000 mg per day, such as from about 20 mg to about 2000 mgper day, such as from about 200 mg to about 2000 mg per day. Forexample, oral dosages can be about 2, about 20, about 200, about 400,about 800, about 1200, about 1600 or about 2000 mg per day. It isunderstood that the total amount per day can be administered in a singledose or can be administered in multiple dosing such as twice, three orfour times per day.

For example, a patient can receive between about 2 mg/day to about 2000mg/day, for example, from about 20 to 2000 mg/day, such as from about200 to about 2000 mg/day, for example from about 400 mg/day to about1200 mg/day. A suitably prepared medicament for once a dayadministration can thus contain between about 2 mg and about 2000 mg,such as from about 20 mg to about 2000 mg, such as from about 200 mg toabout 1200 mg, such as from about 400 mg/day to about 1200 mg/day. TheHDAC inhibitors can be administered in a single dose or in divided dosesof two, three, or four times daily. For administration twice a day, asuitably prepared medicament would therefore contain half of the neededdaily dose.

Intravenously or subcutaneously, the patient would receive the HDACinhibitor in quantities sufficient to deliver between about 3 to 1500mg/m² per day, for example, about 3, 30, 60, 90, 180, 300, 600, 900,1200 or 1500 mg/m² per day. Such quantities can be administered in anumber of suitable ways, e.g., large volumes of low concentrations ofHDAC inhibitor during one extended period of time or several times aday. The quantities can be administered for one or more consecutivedays, intermittent days, or a combination thereof per week (7 dayperiod). Alternatively, low volumes of high concentrations of HDACinhibitor during a short period of time, e.g., once a day for one ormore days either consecutively, intermittently, or a combination thereofper week (7 day period). For example, a dose of 300 mg/m² per day can beadministered for 5 consecutive days for a total of 1500 mg/m² pertreatment. In another dosing regimen, the number of consecutive days canalso be 5, with treatment lasting for 2 or 3 consecutive weeks for atotal of 3000 mg/m² and 4500 mg/m² total treatment.

Typically, an intravenous formulation can be prepared which contains aconcentration of HDAC inhibitor of between about 1.0 mg/mL to about 10mg/mL, e.g. 2.0 mg/mL, 3.0 mg/mL, 4.0 mg/mL, 5.0 mg/mL, 6.0 mg/mL, 7.0mg/mL, 8.0 mg/mL, 9.0 mg/mL and 10 mg/mL and administered in amounts toachieve the doses described above. In one example, a sufficient volumeof intravenous formulation can be administered to a patient in a daysuch that the total dose for the day is between about 300 and about 1500mg/m².

In a preferred embodiment, the histone deacetylase (HDAC) inhibitor isPXD-101 and is administered intravenously at a rate of 900 mg/m² every24 hours.

Glucuronic acid, L-lactic acid, acetic acid, citric acid, or anypharmaceutically acceptable acid/conjugate base with reasonablebuffering capacity in the pH range acceptable for intravenousadministration of the HDAC inhibitor can be used as buffers. Sodiumchloride solution wherein the pH has been adjusted to the desired rangewith either acid or base, for example, hydrochloric acid or sodiumhydroxide, can also be employed. Typically, a pH range for theintravenous formulation can be in the range of from about 5 to about 12.A preferred pH range for intravenous formulation wherein the HDACinhibitor has a hydroxamic acid moiety, can be about 9 to about 12.Consideration should be given to the solubility and chemicalcompatibility of the HDAC inhibitor in choosing an appropriateexcipient.

Subcutaneous formulations, preferably prepared according to procedureswell known in the art at a pH in the range between about 5 and about 12,also include suitable buffers and isotonicity agents. They can beformulated to deliver a daily dose of HDAC inhibitor in one or moredaily subcutaneous administrations, e.g., one, two, or three times eachday. The choice of appropriate buffer and pH of a formulation, dependingon solubility of the HDAC inhibitor to be administered, is readily madeby a person having ordinary skill in the art. Sodium chloride solutionwherein the pH has been adjusted to the desired range with either acidor base, for example, hydrochloric acid, or sodium hydroxide, can alsobe employed in the subcutaneous formulation. Typically, a pH range forthe subcutaneous formulation can be in the range of from about 5 toabout 12. A preferred pH range for subcutaneous formulation wherein theHDAC inhibitor has a hydroxamic acid moiety, can be about 9 to about 12.Consideration should be given to the solubility and chemicalcompatibility of the HDAC inhibitor in choosing an appropriateexcipient.

The HDAC inhibitors can also be administered in intranasal form viatopical use of suitable intranasal vehicles, or via transdermal routes,using those forms of transdermal skin patches well known to those ofordinary skill in that art. To be administered in the form of atransdermal delivery system, the dosage administration will, or course,be continuous rather than intermittent throughout the dosage regime.

The HDAC inhibitors can be administered as active ingredients inadmixture with suitable pharmaceutical diluents, excipients or carriers(collectively referred to herein as “carrier” materials) suitablyselected with respect to the intended form of administration, that is,oral tablets, capsules, elixers, syrups, and the like, and consistentwith conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet orcapsule, the HDAC inhibitor can be combined with an oral, non-toxic,pharmaceutically acceptable, inert carrier such as lactose, starch,sucrose, glucose, methyl cellulose, microcrystalline cellulose, sodiumcroscarmellose, magnesium stearate, dicalcium phosphate, calciumsulfate, mannitol, sorbitol, and the like or a combination thereof. Fororal administration in liquid form, the oral drug components can becombined with any oral, non-toxic, pharmaceutically acceptable inertcarrier such as ethanol, glycerol, water, and the like. Moreover, whendesired or necessary, suitable binders, lubricants, disintegratingagents, and coloring agents can also be incorporated into the mixture.Suitable binders include starch, gelatin, natural sugars such as glucoseor beta-lactose, corn-sweeteners, natural and synthetic gums such asacacia, tragacanth, or sodium alginate, carboxymethylcellulose,microcrystalline cellulose, sodium croscarmellose, polyethylene glycol,waxes and the like. Lubricants that may be used in these dosage formsinclude sodium oleate, sodium stearate, magnesium stearate, sodiumbenzoate, sodium acetate, sodium chloride, and the like. Disintegratorsinclude, without limitation, starch methyl cellulose, agar, bentonite,xanthan gum, and the like.

Suitable pharmaceutically acceptable salts of the histone deacetylaseinhibitors described herein that are suitable for use in the method ofthe invention are conventional non-toxic salts and can include a saltwith a base or an acid addition salt such as a salt with an inorganicbase, for example, an alkali metal salt (e.g., lithium salt, sodiumsalt, potassium salt, etc.), an alkaline earth metal salt (e.g., calciumsalt, magnesium salt, etc.), an ammonium salt; a salt with an organicbase, for example, an organic amine salt (e.g., triethylamine salt,pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt,dicyclohexylamine salt, N,N′-dibenzylethylenediamine salt, etc.) etc.;an inorganic acid addition salt (e.g., hydrochloride, hydrobromide,sulfate, phosphate, etc.); an organic carboxylic or sulfonic acidaddition salt (e.g., formate, acetate, trifluoroacetate, maleate,tartrate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, etc.);a salt with a basic or acidic amino acid (e.g., arginine, aspartic acid,glutamic acid, etc.); and the like.

Combination Therapy

The treatment procedures can take place sequentially in any order,simultaneously, or a combination thereof. For example, the firsttreatment procedure, administration of a HDAC inhibitor, can take placeprior to (e.g., up to 7, 14, 21 days prior to) the second treatmentprocedure, administration of a chemotherapeutic agent or EGFR inhibitor,after (e.g., up to 7, 14, 21 days after) the second treatment procedure,at the same time as the second treatment procedure, or a combinationthereof. In one embodiment, at least a part of the first treatmentprocedure is concurrent with a part of the second treatment procedure.

For example, a total treatment period can be decided for the HDACinhibitor. The chemotherapeutic agent or EGFR inhibitor can beadministered prior to onset of treatment with the HDAC inhibitor orfollowing treatment with the HDAC inhibitor. In addition, thechemotherapeutic agent or EGFR inhibitor can be administered during theperiod of HDAC inhibitor administration but does not need to occur overthe entire HDAC inhibitor treatment period.

In one embodiment, the present invention provides a method of treatingcancer (e.g., multiple myeloma), wherein said method comprisesadministering to a patient, PXD-101 for several days prior to theadministration of dexamethasone (DEX).

In one embodiment, the combination therapy is on a 21-day cycle whereinPXD-101 is administered every 24 hours for five days, then dexamethasone(DEX) and PXD-101 are administered on days 2-5 and 10-13.

In another embodiment, the present invention provides a method oftreating cancer (e.g., epithelial ovarian cancer), wherein said methodcomprises administering to a patient, PXD-101 for several days prior toadministering the carboplatin/paclitaxel regimen. In a specificembodiment, PXD-101 will be administered every 24 hours for 5 days everythree weeks. In each cycle, the carboplatin/paclitaxel regimen will beadministered on cycle day 3 after administration of PXD-101.

In another embodiment, the present invention provides a method oftreating cancer (e.g., lymphoma), wherein said method comprisesadministering to a patient, PXD-101 in a first dose or amount and 5-FUin a second dose or amount, to provide therapeutically effectiveanti-cancer effects.

The histone deacetylase (HDAC) inhibitors and chemotherapeutic agentsand EGFR inhibitors can also be used in a method of inhibiting cellproliferation in a cell comprising contacting the cell with a firstamount of a compound capable of inhibiting a histone deacetylase (e.g.,PXD-101) or a salt or solvate thereof, and contacting the cell with asecond amount of a chemotherapeutic agent or EGFR inhibitor, to prevent,inhibit (fully or partially), or arrest cell proliferation. The cell canbe a transgenic cell. In another embodiment the cell can be in apatient, such as a mammal, for example a human.

In one embodiment, the first amount to treat cancer in a cell is acontact concentration of HDAC inhibitor from about 1 μM to about 50 μMsuch as, from about 1 μM to about 5 μM, for example, from about 1 μM toabout 500 nM, such as from about 1 μM to about 50 mM, for example, 1 μMto about 500 μM. In a particular embodiment, the concentration is lessthan about 5.0 μM. In another embodiment, the concentration is about 500nM.

Kits

One aspect of the invention pertains to a kit or kit-of-partscomprising:

-   -   (a) a histone deacetylase (HDAC) inhibitor, such as PXD-101,        preferably as a component of a pharmaceutically acceptable        formulation, and preferably provided in a suitable container        and/or with suitable packaging; and    -   (b) another chemotherapeutic agent, such as dexamethasone or        5-fluorouracil, or an epidermal growth factor receptor (EGFR)        inhibitor, such as Tarceva®, preferably as a component of a        pharmaceutically acceptable formulation, and preferably provided        in a suitable container and/or with suitable packaging;    -   wherein said kit or kit-of-parts is suitable for use in a method        for treating cancer.

In one embodiment, the kit or kit-of-parts further comprisesinstructions, e.g., written instructions, for use, for example,instructions for administration, e.g., of the two drugs. In oneembodiment, the instructions include a list of indications (e.g.,cancer, types of cancer) for which combination of drugs is a suitabletreatment.

In one embodiment, the kit or kit-of-parts further comprises appropriatereagents (e.g., buffers, solvents) and devices (e.g., tubes, syringes)for assembly and use (e.g., administration).

EXAMPLES

The following examples more fully illustrate the preferred embodimentsof the invention. They should in no way be construed, however, aslimiting the broad scope of the invention, as described herein.

Example 1

The effects of HDAC inhibitors in combination with standardchemotherapeutic agents were tested in vitro to determine the potentialclinical use of an HDAC inhibitor (i.e., PXD-101), in combinationchemotherapy. See, for example, the Data Annexes below.

WST-1 proliferation assays were used to assess the anti-proliferativeeffects of drug combinations. For example, four myeloma cell lines(e.g., JJN3, LP-1, RPMI-8226, U266), the HCT116 human colon carcinomacell line, and the ovarian cell line, A2780, and its platinum-resistantsubline, A2780/cp7, among others, were used in the combination studies.A commercial program, CalcuSyn®, was employed to determine whether thecombined effects are synergistic, antagonistic, or additive.

WST-1 Assay

Cells were cultured, exposed to PXD-101 alone or in combination with achemotherapeutic agent, and incubated for a time, and the number ofviable cells was then assessed using the Cell Proliferation ReagentWST-1 from Boehringer Mannheim (Cat. No. 1 644 807), described below.

Cells were plated in 96-well plates at 3-10×10³ cells/well in 100 μL ofculture medium. The following day, different concentrations of PXD-101alone or in combination with a chemotherapeutic agent were added and thecells incubated at 37° C. for 48 hours. Subsequently, 10 μL/well ofWST-1 reagent was added and the cells re-incubated for 1 hour. After theincubation time, absorbance was measured.

WST-1 is a tetrazolium salt which is cleaved to formazan dye by cellularenzymes. An expansion in the number of viable cells results in anincrease in the overall activity of mitochondrial dehydrogenases in thesample. This augmentation in the enzyme activity leads to an increase inthe amount of formazan dye formed, which directly correlates to thenumber of metabolically active cells in the culture. The formazan dyeproduced is quantified by a scanning multi-well spectrophotometer bymeasuring the absorbance of the dye solution at 450 nm wavelength(reference wavelength 690 nm).

Percent activity (% activity) in reducing the number of viable cells wascalculated for each test compound as:% activity={(S ^(c) −B)/(S ^(o) −B)}×100wherein S^(c) denotes signal measured in the presence of the compoundbeing tested, S^(o) denotes signal measured in the absence of thecompound being tested, and B denotes the background signal measured inblank wells containing medium only. The IC50 corresponds to theconcentration which achieves 50% activity. IC50 values were calculatedusing the software package Prism® 3.0 (GraphPad Software Inc., SanDiego, Calif., USA), setting top value at 100 and bottom value at 0.

Measurement of cell viability in the presence of increasingconcentration of test compound at different time points is used toassess both cytotoxicity and the effect of the compound on cellproliferation.

The results were further analyzed using the Combination Index (CI)method with the CalcuSyn® program from Biosoft. A CI value of less than1 indicates synergy; a CI value of 1 indicates an additive effect; and aCI value of greater than 1 indicates antagonism.

Using the CalcuSyn® program, the CI is determined by the isobologramequation CI=(D)1/(Dx)1+(D)2/(Dx)2. Drug 1 (D1) and drug 2 (D)2 incombination inhibit X % and (Dx)1 and (Dx)2 are the doses of drug 1 anddrug 2 alone that also inhibits X5. For each compound, the % growthvalues at each dose as determined in the WST-1 assay is used. CI valuesthat are less than 1, equal to 1, or are greater than 1 indicatesynergism, additive effect, or antagonism, respectively. CI's arecompared at various percent inhibitory concentrations.

Results

PXD-101+5-FU

The anti-proliferative effects of combining PXD-101 and 5-FU wereexamined in a variety of cell lines (e.g., HT-116, A2780) and undervarying conditions, e.g., simultaneously, 24 hours before, or 24 hoursafter administration of the chemotherapeutic agent 5-FU. The results ofthese experiments are summarized in FIG. 2 and Data Annex 3 which showthe synergy of PXD-101 and 5-FU in the WST-1 anti-proliferative assay ascalculated by CalcuSyn®.

FIG. 2 and Data Annex 5 depicts a graph and chart, respectively, showingthe synergy of PXD-101 and 5-FU in a WST-1 anti-proliferative assay ascalculated by CalcuSyn®.

To determine whether the combinations are additive, synergistic, orantagonistic, isobolograms are plotted and combination indicescalculated using the commercial software program CalcuSyn®. Inisobolograms, the X intercepts indicate the concentrations of one drugwhich results in a given percentage of growth inhibition and the Yintercepts indicate the concentrations at which the other drug inhibitedcell growth. The data point that falls between the axes indicates theconcentration of the drug combination that inhibits cell growth. Thefurther above or below this data point deviates from the straight linejoining the intercept, the more antagonistic or synergistic the effect,respectively. Combination data points that fall on or close to the linejoining the intercepts indicates additive effects.

FIG. 1 depicts an isobologram for the combination of PXD-101 and 5-FU ina HCT116 cell line, wherein 5-FU was added alone 24 hours after PXD-101.The graph demonstrates that this combination produces a surprisinglysynergistic effect.

In the lymphoma cell line, a synergistic effect was observed when 5-FUwas added 24 hours after the administration of PXD-101.

Data Annex 8 shows that a synergy between PXD-101 and 5-FU exists alsoin vivo. In the Mouse P388 leukemia model, the survival of the micetreated with the drug combination is greater than the survival of thosetreated with each drug alone. Notably, there is also a synergy when miceare treated with PXD-101 and a combination of 5-FU and oxaliplatin (seeData Annex 9).

PXD-101 Fludarabine

There is a synergistic effect of PXD-101 and fludarabine in the mouseP388 model: the survival of the mice treated with the combination ofdrugs is greater than the survival of mice treated with each drug alone(see Data Annex 10).

PXD-101 and Other Chemotherapeutic Agents

L-phenylalanine mustard (PAM, melphalan) shows synergy with PXD-101 in anumber of myeloma cell lines (see Data Annex 1). Furthermore, synergycan be seen between PXD-101 and other chemotherapeutic agents,specifically cisplatin, 5-FU, topotecan, gemcitabine, docetaxel,doxorubicin, tamoxifen, dexamethasone, and 5-azacytidine, in a number oftumour cell lines (see Data Annex 2). Finally, synergy of PXD-101 withchlorambucil, and synergy of PXD-101 with fludarabine, is seen in cellproliferation experiments in cultured lymphoma cells (see Data Annex 4).

PXD-101+Dexamethasone

The anti-proliferative effects of adding PXD-101 simultaneously (e.g.,48 hour co-incubations), 24 hr after, or 24 hr before adding thechemotherapeutic agent, dexamethasone to the JJN3, LP-1, RPMI-8226, U266tumor cell lines were also examined by the methods described above.

When cells were treated with PXD-101 in combination with dexamethasonesimultaneously (i.e., at 48 hour co-incubations), 24 hour PXD-101 then48 hour PXD-101+DEX, or 24 hour DEX, then 48 hour PXD-101+DEX,synergistic effects were obtained for the LP-1, RPMI-8226, and U266tumor cell lines over a range of concentrations (see Data Annex 1).Strong synergy (CI<0.3) was observed in the U266 cell line when PXD-101was administered for 24 hours, followed by 48 hour PXD-101+DEX, and inthe LP1 cell line when DEX was administered for 24 hours, followed by48-hour co-incubation of PXD-101 and DEX over a range of concentrationsand conditions (see Data Annex 1).

PXD-101+Doxorubicin

The myeloma cell lines (e.g., JJN3, LP-1, RPMI-8226, U266) were treatedwith PXD-101 and doxorubicin. As described above, the combined effectsof these compounds on the myeloma cell lines was determined by plottingisobolograms and calculating combination indices.

Additive to synergistic effects were observed for this combination whenPXD-101 and doxorubicin were co-incubated for 48 hours over a range ofconcentrations (see Data Annex 1 and Data Annex 2).

PXD-101+Vincristine

The myeloma cell lines (e.g., JJN3, LP-1, RPMI-8226, U266) were treatedwith PXD-101 and vincristine. As described above, the combined effectsof these compounds on the myeloma cell lines was determined by plottingisobolograms and calculating combination indices. Additive tosynergistic effects were observed for this combination when PXD-101 andvincristine were co-incubated over a range of concentrations (see DataAnnex 1).

PXD-101+Alimta®

The antifolate pemetrexed (Alimta®) is marketed for use against lungcancers. There is a clear synergistic effect of combining PXD-101 withAlimta® in proliferation experiments using cultured cells (see DataAnnex 12 and FIG. 6).

Discussion

This example reports the effect of combining PXD-101 with standardchemotherapeutics. There is strong synergy between PXD-101 and DEX whenU266 cells are incubated with PXD-101 for 24 hours prior to theadministration of DEX, and when LP1 cells are incubated with DEX for 24hours prior to the administration of PXD-101 over a range ofconcentrations and conditions (See Data Annex 1 and FIG. 1).

Combination of PXD-101 with doxorubicin on myeloma cell lines producedadditive to synergistic effects when the compounds were co-incubatedover a range of concentrations and conditions (see Data Annex 1).

Similarly, combination of PXD-101 with cisplatin on ovarian cell linesproduced additive to synergistic effects when the compounds wereco-incubated over a range of concentrations and conditions (see DataAnnex 2).

Moreover, combination of PXD-101 with 5-FU produced strong synergism ina variety of cell lines (e.g., ovarian cell lines, A2780 and OVCAR3,lung cell line, NYH, breast cancer cell line, MCF-7, and colon cancercell lines, HT116 and HT116p53). In most circumstances, the strongestsynergism was observed when 5-FU was added 24 hours after theadministration of PXD-101. (See Data Annex 2.) In addition, thecombination of PXD-101 and 5-FU produced synergistic effects in thelymphoma cell line when 5-FU was administered 24 hours after theadministration of PXD-101.

Accordingly, the above combination studies identify effectivetherapeutic combinations for use in clinical trials of PXD-101.

Example 2 Clonogenic Assay

A clonogenic assay was performed essentially as described (Jensen etal., 1993) to determine the anti-proliferative effects of combinationsinvolving PXD-101, in particular a combination involving PXD-101 and5-FU.

In this assay, HCT116 or p388 cells were exposed to combinations ofPXD-101 and 5-FU for the indicated times. Cells were then plated in 0.3%agar in 6 cm petri dishes with sheep red blood cells as feeder layer intriplicate, and were incubated at 37° C. Plates were counted after 14-21days. A summary of the results is provided in Data Annex 6. Data Annex 7and FIG. 3 present the data underlying this summary, and shows thesynergy of PXD-101 and 5-FU in the clonogenic assay as calculated byCalcuSyn®.

Data Annex 6 demonstrates that combinations of PXD-101 and 5-FU aresynergistic to strongly synergistic in both cell lines when PXD-101 and5-FU were co-incubated for 24 hours and when PXD-101 was administered 24hours after the administration of 5-FU. The data also demonstrate thatthis combination produces strong synergistic effects when 5-FU isadministered 24 hours after the administration of PXD-101.

Example 3 In Vivo Studies

To investigate the tumour growth inhibitory effect of PXD-101 in aHCT116 colon cancer subcutaneous xenograft model (nu/nu mice), twoPXD-101 doses were included, 60 and 100 mg/kg scheduled q1d×5/week for 2weeks. The chemo-sensitising effect of PXD-101 in combination with 5-FUwas also investigated. The 5-FU dose was set at 15 mg/kg.

PXD101 caused significant tumour growth inhibition in a HCT-116 coloncancer xenograft model in nude mice. 5-FU was ineffective in the HCT116model in the chosen dose regimen, which indicates suboptimal dosing.

FIG. 4 shows the chemo-sensitising effect of PXD101. HCT116 colon cancerxenograph. Daily i.p. treatments with PXD101 (morning) and 5-FU(afternoon). Mean/SEM, n=10(9).

Chemo-sensitising effect of PXD101 on 5-FU treatment was indicated. Theresults indicated synergistic effect of PXD101 and 5-FU after the firsttreatment cycle where single 5-FU therapy is ineffective at 15 mg/kg.FIG. 4 depicts a graph summarizing the chemo-sensitizing effects ofPXD-101 in a HCT116 colon cancer xenograft.

P388 Mouse Model

P388 mouse leukemia cells were injected intraperitoneally (IP) intoB6D1F1 female mice. PXD-101 and/or chemotherapeutic agent wereadministered IP on day 3, then daily for the required number ofconsecutive days. Kaplan-Meier survival distribution graphs are shownfor each combination.

Example 4 Growth Inhibition Assays Involving EGFR Inhibitors and HDACInhibitors

The objective of this study was to determine whether PXD-101 monotherapyor combination therapy with Tarceva® effects the growth of A431 cells(human epidermoid carcinoma) or Calu-3 cells (human non-small cell lungcancer).

Materials and Methods:

Cells were plated at 3,000 cells/well in 96-well plates, treated withPXD-101 or Tarceva® (an EGFR-kinase inhibitor) alone or in combinationat the indicated concentrations for 72 hours, and evaluated forgrowth/viability via the CellTiter-Glo assay described above.

Results:

The combination of PXD-101+Tarceva® causes greater growth-inhibitionthan either drug used alone. See, for example, FIG. 5 and Data Annex 11.This effect is synergistic.

Conclusion:

PXD-101 used in combination with Tarceva® may be beneficial for thetreatment of non-small cell lung cancer and perhaps other types ofcancers.

Example 5 Western Blot

The objective of this study was to determine whether PXD-101 treatmenteffects EGFR protein levels.

Materials and Methods:

A431 cells (human epidermoid carcinoma) were plated at ˜80% confluencyin 6-well plates, treated with PXD-101 at the indicated concentrationsfor 24 hours, and then whole-cell lysates were harvested and analyzed byWestern blotting using an anti-EGFR antibody. The blot was reprobed withan anti-actin antibody to control for lane loading.

Results:

PXD-101 treatment apparently decreases the level of EGFR protein in A431cells.

Conclusion:

A431 cells are known to overexpress wild-type EGFR and to be sensitiveto growth-inhibition by reagents that inhibit the EGFR pathway. Thereduction of EGFR protein induced by PXD-101 may be one mechanism bywhich PXD-101 used alone is growth-inhibitory to these cells. Moreover,the PXD-101-mediated reduction in EGFR protein may provide a mechanisticrationale for the additive/synergistic growth-inhibition observed whenPXD-101 is used in combination with Tarceva®.

Additional Data Data Annex 1

Summary of MYELOMA combination studies with PXD101 in vitro. Numbers inbrackets is a guide to the schedule used in each experiment (see below).Results in Table are those produced using “standard” combinationprotocol.

PXD101 Combined With: Cell Line Vincristine Doxorubicin PAMDexamethasone JJN3 −(1), +(2), +(1), −(2,3) +(1,2), −(2) ++(3) ++(3)LP-1 −(2), +(1,3) ++(1), −(2,3) −(1,3), +(2) ++(1), ++(2), +++(3) RPMI-++(1), −(2,3) +(1,2), ++(3) +(1,2), +(2) 8226 ++(3) U266 +(1), −(2,3)++(1), +(2), −(3) +(1,3), +++(2), ++(3) ++(2) At least three points fallon or below CI value corresponding to: CI < 0.3 very synergistic +++ CI0.3-0.7 synergistic ++ CI 0.7-1 additive + CI > 1 antagonistic — notdone nd (1) 48 hour co-incubations (2) 24 hour PXD101 then 48 hourPXD101 & compound (3) 24 hour compound then 48 hour PXD101 & compound

JJN3, RPMI 8226 & U266 cells are dexamethasone resistant.

Data Annex 2

Summary of the combination studies with PXD101 in vitro. Numbers inbrackets is a guide to the schedule used in each experiment (see below).

PXD101 Combined With: Cell Line Cisplatin 5-FU Topotecan GemcitabineA2780 ++(1,3,4,7,8,12) +++(3,1) ++(1) ++(1) (ovarian) A2780cp70 ++(1) ndnd nd (ovarian) ++(4,7,8,12) OVCAR3 ++(1,4,8,12), +++(1,7), nd nd(ovarian) +++(7) ++(11), +(3) PC-3 ++(1,4), ++(4) +(1) ++(1) (prostate)+++(7,8,12) NYH −(1,4,8,12), +++(4) +(1) ++(2) (lung) +(7) MIA-PA-CA2 ndnd nd −(6), (pancreatic) −(4) P-388 −(1), −(7), −(1), +(1,8) (leukaemic)++(4,8) +(1,3,8), +(8), ++(10) ++(4) MCF-7 +(8), ++(5) ++(1) +(4,6)(breast) ++(1,4,7,12) +++(4) T47-D nd nd nd nd (breast) MDA-MB-231 nd ndnd nd (breast) HT-29 +++(1,7), −(1), +(4) +(1) ++(2) (colon) ++(4,8),++(3,5,8,9), +(12) +++(7) HT116 nd ++(1,11), nd nd (colon) +++(7)HT116CMV nd ++(1,7,11) nd nd (colon) HT116p53 nd ++(1,11), nd nd (colon)+++(7) LNCaP nd nd nd nd (prostate) PXD101 Combined With: Cell LineDocetaxel Doxorubicin Tamoxifen Dexamethasone A2780 nd nd nd nd(ovarian) A2780cp70 nd nd nd nd (ovarian) OVCAR3 −(1,7,8), nd nd nd(ovarian) +(4,12) PC-3 +(1), −(1) nd −(1,2,14), (prostate) ++(3) +(13)NYH nd nd nd nd (lung) MIA-PA-CA2 nd nd nd nd (pancreatic) P-388 nd−(1), nd nd (leukaemic) +(8) MCF-7 nd −(1,7) −(1,3,11), nd (breast)+(12) T47-D nd +(1,7) +(1,3,11,12) nd (breast) MDA-MB-231 nd +++(1)−(3), nd (breast) +(11), ++(1) HT-29 ++(1) nd nd nd (colon) HT116 nd ndnd nd (colon) HT116CMV nd nd nd nd (colon) HT116p53 nd nd nd nd (colon)LNCaP nd nd nd −(3) (prostate) PXD101 Combined With: Cell Line5-Azacytidine Alimta ® Irinotecan Oxaliplatin A2780 nd nd nd nd(ovarian) A2780cp70 nd nd nd nd (ovarian) OVCAR3 nd nd nd nd (ovarian)PC-3 nd nd nd nd (prostate) NYH nd nd nd nd (lung) MIA-PA-CA2 nd nd ndnd (pancreatic) P-388 nd nd nd nd (leukaemic) MCF-7 ++(1,4,8) nd nd nd(breast) T47-D +(7), ++(1,4), nd nd nd (breast) +++(8), MDA-MB-231++(1,4,7) nd nd nd (breast) +(8,11), −(15) HT-29 nd nd nd nd (colon)HT116 nd −(7), −(1,3), +(2), −(1), (colon) +(1,2,3,4) ++(4,7,12)+(2,3,4,7,12) ++(12) HT116CMV nd nd nd nd (colon) HT116p53 nd nd nd nd(colon) LNCaP nd nd nd nd (prostate) At least three points fall on orbelow CI value corresponding to: CI <0.3 very synergistic +++ CI 0.3-0.7synergistic ++ CI 0.7-1 additive + CI >1 antagonistic — not done nd (1)48 hour co-incubations (2) 72 hour co-incubations (3) 24 hour PXD101then 24 hour PXD101 & compound (4) 24 hour compound then 48 hour PXD101& compound (5) 48 hour compound then 48 hour PXD101 & compound (6) 96hour co-incubations (7) 24 hour PXD101 then 48 hour compound alone (8)24 hour PXD101 then 48 hour PXD101 & compound (9) 24 hour PXD101 then 72hour compound alone (10) 24 hour compound then 24 hour PXD101 & compound(11) 24 hour PXD101 then 24 hour compound alone (12) 24 hour compoundthen 48 hour PXD101 alone (13) 96 hour co-incubations (14) 48 hourPXD101 then 72 hour compound alone (15) 6 hour PXD101 then 24 hourcompound

Data Annex 2 (Continued) Data Annex 2 (Continued) Data Annex 3

PXD-101 + 5-FU Cell Line Combined Effect A2780 (ovarian) +++(3,1) OVCAR3(ovarian) +++(1,7), +(11), +(3) PC-3 (prostate) ++(4) NYH (lung) +++(4)P-388 (leukaemic) −(7), +(1,3,8), ++(10) MCF-7 (breast) ++(5), +++(4)HT-29 (colon) −(1), +(4), ++(3,5,8,9), +++(7) HT116 (colon) ++(1,11),+++(7) HT116CMV (colon) ++(1,7,11) HT116p53 (colon) ++(1,11), +++(7) Atleast three points fall on or below Cl value corresponding to: CI < 0.3very synergistic +++ CI 0.3-0.7 synergistic ++ CI 0.7-1 additive + CI >1 antagonistic — (1) 48 hour co-incubations (2) 72 hour co-incubations(3) 24 hour PXD101 then 24 hour.PXD101 & 5-FU (4) 24 hour compound then48 hour PXD101 & 5-FU (5) 48 hour compound then 48 hour PXD101 & 5-FU(6) 96 hour co-incubations (7) 24 hour PXD101 then 48 hour FU-5 alone(8) 24 hour PXD101 then 48 hour PXD101 & FU-5 (9) 24 hour PXD101 then 72hour FU-5 alone (10) 24 hour compound then 24 hour PXD101 & FU-5 (11) 24hour PXD101 then 24 hour FU-5 alone

Data Annex 4

Summary of LYMPHOMA combination studies with PXD101 in vitro. Numbers inbrackets are a guide to the schedule used in each experiment (seebelow). Results in Table are those produced using “standard” combinationprotocol.

PXD combined with: Cell Line Chlorambucil Fludarabine SU-DHL-4 −(1,3),++(2) −(2), +(l), ++(3) At least three points fall on or below CI valuecorresponding to: CI < 0.3 very synergistic +++ CI 0.3-0.7 synergistic++ CI 0.7-1 additive + CI > 1 antagonistic — not done nd (1) 48 hourco-incubations (2) 24 hour PXD101 then 48 hour PXD101 & compound (3) 24hour compound then 48 hour PXD101 & compound

Data Annex 5

WST-1 Assay Combination No. PXD-101 (μM) 5-FU (μM) Combination Index 10.187 2.22 1.253 2 0.28 3.33 0.429 3 0.42 5 0.307 4 0.63 7.5 0.222 50.945 11.25 0.212 6 1.42 16.88 0.265 7 2.13 25.31 0.375 8 3.195 37.970.462

Data Annex 6

Summary of Clonogenic Assay combination studies with PXD101/5-FU invitro.

24h co- 24h PXD101 + 24h 5-FU + Cell Line incubations 24h 5-FU 24hPXD101 P-388 ++ + ++ HCT-116 ++ +++ +++ At least three points fall on orbelow CI value corresponding to: CI < 0.3 very synergistic +++ CI0.3-0.7 synergistic ++ CI 0.7-1 additive + CI > 1 antagonistic — notdone nd

Data Annex 7

Clonogenic Assay Combination No. PXD-101 (μM) 5-FU (μM) CombinationIndex 1 100 135 0.16 2 50 90 0.416 3 25 60 0.538 4 12.5 40 0.577 5 6.2526.67 0.823 6 3.125 17.78 1.04 7 1.5626 11.85 0.86 8 0.78125 7.9 0.871

Data Annex 8

In vivo synergy of 5-FU and PXD101 in the P388 mouse model.

Cumulative Survival 5-FU (30) + 5-FU (30) + Time PXD101 PXD101 PXD101PXD101 Vehicle (days) 5-FU (30) (100) (60) (100) (60) control 9 1 1 1 11 0.70 10 1 1 1 1 1 0.43 11 1 1 1 0.85 1 0.15 12 1 1 1 0.43 0.70 0 13 11 1 0.43 0.15 — 14 1 1 1 0.15 0 — 15 0.85 1 1 0 — — 16 0.55 1 1 — — — 170.15 1 1 — — — 18 0 1 0.85 — — — 19 — 1 0.30 — — — 20 — 0.43 0.15 — — —21 — 0.30 0.15 — — — 22 — 0.15 0.15 — — — 23 — 0 0.15 — — — 24 — — 0 — ——

Data Annex 9

In vivo synergy of PXD 101 with the combination of oxaliplatin and 5-FUin the P388 mouse model.

Cumulative Survival PXD101 (100) + PXD101 (60) + Time 5-FU Oxali Oxali(5) + Oxali (5) + Oxali (5) + Vehicle (days) (50) (5) 5-FU (50) 5-FU(50) 5-FU(50) control 9 1.00 0.45 1.00 1.00 1.00 0.85 10 0.55 0 0.551.00 1.00 0.75 11 0.15 — 0.30 1.00 1.00 0.45 12 0.15 — 0.30 1.00 0.850.35 13 0.15 — 0.30 1.00 0.85 0.35 14 0.15 — 0.30 1.00 0.85 0.35 15 0.15— 0.30 1.00 0.85 0.20 16 0.15 — 0.30 0.85 0.85 0.15 17 0.15 — 0.30 0.700.70 0.15 18 0.15 — 0.30 0.55 0.45 0.10 19 0.15 — 0.30 0.55 0.30 0.10 200.15 — 0.30 0.55 0.30 0.10 21 0.15 — 0.30 0.55 0.30 0.10 22 0.15 — 0.300.45 0.30 0 23 0.15 — 0.30 0.45 0.30 — 24 0 — 0.30 0.45 0.15 — 25 — —0.30 0.45 0.15 — 26 — — 0.30 0.30 0.15 — 27 — — 0.30 0.30 0.15 — 28 — —0.15 0.15 0.15 — 29 — — 0.15 0.15 0.15 — 30 — — 0.15 0.15 0.15 — 31 — —0 0 0 —

Data Annex 10

In vivo synergy of PXD 101 with fludarabine in the P388 mouse model.

Cumulative Survival Time Fludarabin PXD101 (100) + PXD101 (60) + Vehicle(days) (50) Fludarabin (50) Fludarabin (50) control 8 1.00 1.00 1.000.70 9 0.85 1.00 1.00 0.55 10 0.55 1.00 1.00 0.40 11 0.40 1.00 1.00 0.3012 0.40 1.00 1.00 0.30 13 0.40 1.00 1.00 0.30 14 0.40 0.50 1.00 0.30 150.30 0.35 0.55 0.30 16 0.30 0.17 0.55 0.15 17 0.15 0 0.30 0 18 0.15 —0.30 — 19 0 — 0.15 — 20 — — 0 —

Data Annex 11

Data from proliferation experiments using cultured cells, demonstratingthe synergy of PXD101 with Tarceva®.

Calu-3 (wtEGFR; moderately responsive to Tarceva ®) PXD101 (μM)Tarceva ® (μM) Combination Index 0.0313 0.01 0.556 0.0625 0.0316 0.3710.125 0.1 0.479 0.25 0.316 0.525 0.5 1.0 0.549 1.0 3.16 0.641 HCC-4006(mutant EGFR; strongly responsive to Tarceva ®) PXD101 (μM) Tarceva ®(μM) Combination Index 0.351 0.0391 0.447 0.527 0.078 0.294 0.79 0.1560.154 1.185 0.313 0.166 1.778 0.625 0.245 2.667 1.25 0.309 Range of CIDescription Symbol <0.1 Very strong synergism +++++ 0.1-0.3 Strongsynergism ++++ 0.3-0.7 Synergism +++  0.7-0.85 Moderate synergism ++0.85-0.90 Slight synergism +  0.9-1.10 Nearly additive ± 1.10-1.20Slight antagonism — 1.20-1.45 Moderate antagonism ——

Data Annex 12

Data from proliferation experiments using cultured cells, demonstratingthe synergy of PXD101 with Alimta®.

A549 PDX101 (μM) Alimta (μM) Combination Index 0.351 0.0195 1.339 0.5270.0293 0.694 0.79 0.0439 0.301 1.185 0.0658 0.179 1.778 0.0988 0.1782.667 0.1482 0.231 4.0 0.2222 0.302 6.0 0.3333 0.338 9.0 0.5 0.557NCI-H460 PDX101 (μM) Alimta (μM) Combination Index 0.351 0.0196 0.8930.527 0.0391 0.374 0.79 0.078 0.193 1.185 0.156 0.157 1.778 0.313 0.1612.667 0.625 0.216 4.0 1.25 0.289 6.0 2.5 0.362 9.0 5.0 0.528 Range of CIDescription Symbol <0.1 Very strong synergism +++++ 0.1-0.3 Strongsynergism ++++ 0.3-0.7 Synergism +++  0.7-0.85 Moderate synergism ++0.85-0.90 Slight synergism +  0.9-1.10 Nearly additive ± 1.10-1.20Slight antagonism — 1.20-1.45 Moderate antagonism ——

Data Annex 13

In the following table, the median survival time for mice treated withPXD101 and 5-FU, alone or in combination, is listed together withminimum and maximum observed survival time within each group.

The percentage Increased Life Span (ILS %) was calculated as the mediansurvival (treatment group) minus median survival (vehicle control)divided by median survival (vehicle control) multiplied with 100:

${{ILS}\mspace{14mu}\%} = {\frac{\begin{matrix}{{{Median}\mspace{14mu}{survival}\mspace{14mu}\left( {{treatment}\mspace{14mu}{group}} \right)} -} \\{{Median}\mspace{14mu}{survival}\mspace{14mu}\left( {{vehicle}\mspace{14mu}{control}} \right)}\end{matrix}}{{Median}\mspace{14mu}{survival}\mspace{14mu}\left( {{vehicle}\mspace{14mu}{control}} \right)} \times 100}$

The effect of PXD101 and 5-FU combined treatment was compared to 5-FUtreatment alone using Log Rank statistic analysis and p-values <0.05 areconsidered statistical significant.

Survival time Median Treatment survival Min Max N ILS % P-value*Untreated control 10 9 11 10 Vehicle control 10 9 12 11 5-FU(30) 16 1518 12 60.0% PXD101(60) 13 12 14 7 30.0% PXD101(100) 12 11 14 7 20.0%PXD101(30) + 19 17 19 5 90.0% 0.022 5-FU(30) PXD101(60) + 19 18 24 1290.0% <0.0001 5-FU(30) PXD101(100) + 5- 21 10 24 13 110.0% <0.0001FU(30) P388 i.p. mouse leukemia tumour. Intraperitoneal treatment withPXD101 (mg/kg/treat) and 5-FU (mg/kg/treat) i.p. daily for 5 daysstarting day 3 after tumour implantation. ILS % percentage IncreasedLife Spand. *Log Rank statistic: Compared to 5-FU (30 mg/kg) alone.

When P388 IP implanted mice were treated with PXD101 and 5-FU (MTD) incombination significantly prolonged survival was observed compared towhen PXD101 or 5-FU was given as single treatment. A dose responseeffect of the PXD101 when added to the 5-FU treatment was indicated.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

Thus, while the preferred embodiments of the invention have beenillustrated and described, it is to be understood that this invention iscapable of variation and modification, and should not be limited to theprecise terms set forth. The inventors desire to avail themselves ofsuch changes and alterations which may be made for adapting theinvention to various usages and conditions. Such alterations and changesmay include, for example, different pharmaceutical compositions for theadministration of the agents according to the present invention to amammal; different amounts of agent in the compositions to beadministered; different times and means of administering the agentsaccording to the present invention; and different materials contained inthe administration dose including, for example, combinations ofdifferent agents, or combinations of the agents according to the presentinvention together with other biologically active compounds for thesame, similar or differing purposes than the desired utility of thoseagents specifically disclosed herein. Such changes and alterations alsoare intended to include modifications of the specific desired agentsdescribed herein in which such changes alter the agent in a manner asnot to change the desired potential of the agent, but as to changesolubility of the agent in the pharmaceutical composition to beadministered or in the body, absorption of the agent by the body,protection of the agent for either shelf life or within the body untilsuch time as the biological action of the agent is able to bring aboutthe desired effect, and such similar modifications. Accordingly, suchchanges and alterations are properly intended to be within the fullrange of equivalents, and therefore within the purview of the followingclaims.

The invention and the manner and process of making and using it havebeen thus described in such full, clear, concise and exact terms so asto enable any person skilled in the art to which it pertains, or withwhich it is most nearly connected, to make and use the same.

The invention claimed is:
 1. A method for treating multiple myelomacomprising administering to a patient in need thereof, a first amount ofa histone deacetylase (HDAC) inhibitor, and a second amount ofdexamethasone, wherein the first and second amounts together comprise atherapeutically effective amount, wherein the HDAC inhibitor is thefollowing compound and pharmaceutically acceptable salts and solvatesthereof


2. The method according to claim 1, wherein the HDAC inhibitor is thefollowing compound: